Biology of chlamydospores of Phytophthora ramorum

Abstract:

The chlamydospore is a survival spore produced by 35 of the 75 described species of Phytophthora. Phytophthora ramorum Werres, de Cock & Man in't Veld, the causal agent of Sudden Oak Death (SOD), produces abundant chlamydospores in artificial culture and plant tissue. The chlamydospore is likely the most important survival structure produced by P. ramorum as it is known to exist in wildland, interface, and nursery environments in the United States and Europe. I documented, in varying degrees and combinations, the biology of the chlamydospore in P. ramorum grown in V8 juice agar and broth (V8JB) and detached Rhododendron macrophyllum (Pacific rhododendron) leaves, related to its development, maturation, and germination. I compared these results to what is known about chlamydospores produced in other Phytophthora species. V8 agar- and V8JB-grown P. ramorum chlamydospores had the third largest diameter (avg. 52.4, max. 93.0 μm) and fourth thickest wall (avg. 2.3, max. 5.0μm) of chlamydospore-producing Phytophthora species. In V8 agar and V8JB, maturity was reached in most chlamydospores by eight to ten days, by which time a septum had formed, chlamydospore expansion had ceased, and thickening of the interior wall had begun. The walls of some chlamydospores continued to thicken (from 0.5 to 4.0 μm) up to 120 days of age in V8JB. Generally, larger diameter chlamydospores tended to have thicker walls in P. ramorum. Ten-day-old V8JB-grown chlamydospores of P. ramorum germinated at low and variable rates after 24 hours of incubation at 20°C in the dark. Germination was significantly higher on nutrient-rich V8 agar (max. 12.9 %, min. 0.2%) than cornmeal agar amended with antibiotics (max. 3.6 %, min. 0.5 %) or water agar (max. 4.0 %, min. 0.3 %), suggesting that germination was stimulated by the presence of exogenous nutrients. Among chlamydospores from populations of ten-day-old V8JB cultures, the chlamydospores with smaller diameters (42.7 μm) and thinner walls (1.6 μm) were more likely to germinate. It was unclear if there was any significant difference in rates of germination among V8JB-grown chlamydospores aged 10, 90, and 120 days. Ten-day-old V8JB-grown chlamydospores continued to germinate after up to five days of incubation on V8 agar amended with antibiotics (V8ARP). Lesions on Pacific rhododendron detached leaves that had been infected 16 days had more chlamydospores (23 / mm²) than two-day-old lesions (1 / mm²) with an estimated total of about 10,000 chlamydospores formed within each full lesion. Mean chlamydospore diameter and wall thickness of 30-day-old leaf-grown chlamydospores
was 47.6 μm and 2.9 μm respectively; this was smaller and thicker than those produced on V8 agar (diameter 50.8 μm , wall thickness 2.1 μm) or V8JB-grown (diameter 54.0 μm, wall thickness 2.1 μm) chlamydospores the same age. The maximum chlamydospore wall thickness was also much thicker in leaf-grown chlamydospores (maximum 8.0 μm) than V8 agar (maximum 4.8 μm) or V8JB-grown (maximum 5.0 μm) chlamydospores. Extracted leaf-grown chlamydospores germinated and formed new colonies at a low frequency over 12 days of incubation on V8ARP. P. ramorum was recovered from 100 % of freshly plated leaf lesions, but no recovery of the fungus was seen on cornmeal agar amended with antibiotics (CARP) after leaf lesions were frozen or air-dried for five or ten days. Chlamydospores extracted from leaf lesions frozen 15 days failed to germinate on V8ARP. The experiments presented here represent an important first step in understanding the biology of P. ramorum chlamydospores. Further experimentation utilizing these results may lead to effective disease mitigating measures, but more research is necessary to attain this important goal.